Long non-coding RNA MLLT4 antisense RNA 1 induces autophagy to inhibit tumorigenesis of cervical cancer through modulating the myosin-9/ATG14 axis

The regulatory mechanism of long non-coding RNAs (lncRNAs) in autophagy is as yet not well established. In this research, we show that the long non-coding RNA MLLT4 antisense RNA 1 (lncRNA MLLT4-AS1) is induced by the MTORC inhibitor PP242 and rapamycin in cervical cells. Overexpression of MLLT4-AS1 promotes autophagy and inhibits tumorigenesis and the migration of cervical cancer cells, whereas knockdown of MLLT4-AS1 attenuates PP242-induced autophagy. Mass spectrometry, RNA fluorescence in situ hybridization (RNA-FISH), and immunoprecipitation assays were performed to identify the direct interactions between MLLT4-AS1 and other associated targets, such as myosin-9 and autophagy-related 14(ATG14). MLLT4-AS1 was upregulated by H3K27ac modification with PP242 treatment, and knockdown of MLLT4-AS1 reversed autophagy by modulating ATG14 expression. Mechanically, MLLT4-AS1 was associated with the myosin-9 protein, which further promoted the transcription activity of the ATG14 gene. In conclusion, we demonstrated that MLLT4-AS1 acts as a potential tumor suppressor in cervical cancer by inducing autophagy, and H3K27ac modification-induced upregulation of MLLT4-AS1 could cause autophagy by associating with myosin-9 and promoting ATG14 transcription.


MLLT4-AS1 is a cytoplasmic lncRNA and facilitated autophagy in cervical cancer cells
Using RNA-FISH, we proved that MLLT4-AS1 was mainly distributed in the cytoplasm of cervical cancer cells (Fig. 2A).We confirmed this conclusion using cellular fractionation PCR (Fig. 2B), indicating that MLLT4-AS1 regulates downstream pathways at the post-transcriptional level.
To further assess the function of MLLT4-AS1 in autophagy, the data showed that MLLT4-AS1 overexpression significantly increased autophagy flux-the conversion of LC3-I to LC3-II and SQSTM1 degradation in cervical cancer cells (Fig. 2C).Consistently, similar results were obtained by immunofluorescence staining the LC3-II puncta in MLLT4-AS1-overexpressed cervical cancer cells (Fig. 2D).

PP242 increases MLLT4-AS1 expression by inducing H3K27 acetylation
We evaluated the underlying mechanism of MLLT4-AS1 upregulation in PP242-treated cervical cancer cells.By analyzing the transcriptional modification regions in the UCSC Genome Browser (http:// genome.ucsc.edu/), we found an enriched H3K27ac binding area upstream of the MLLT4-AS1 promoter region in seven cell lines (Fig. 3A and Figure S3).Next, a ChIP assay using anti-H3K27ac antibody was performed and observed that H3K27ac was enriched at the MLLT4-AS1 promoter region.To further confirm the effects of PP242 treatment, the results of RIP assay showed that the levels of H3K27AC enrichment were increased in PP242-treated cells compared with control cells (Fig. 3B).Conversely, cervical cancer cells treated with C646 (a histone acetyltransferase inhibitor) had decreased H3K27AC enrichment at MLLT4-AS1 promotor region (Fig. 3C).Consistently, MLLT4-AS1 was also decreased in cervical cancer cells with C646 treatment compared with PBS-treated cells (Fig. 3D).In conclusion, this indicated that autophagy inducer treatment increases the levels of MLLT4-AS1 by inducing H3K27AC enrichment at the promoter region.However, the detailed regulatory mechanism needs further to study. www.nature.com/scientificreports/

MLLT4-AS1 inhibits tumorigenesis and transformation ability of cervical cancer cells
Excessive autophagy may lead to cell death, known as autophagy-dependent cell death (ADCD) 18 .To further detect the role of MLLT4-AS1 in mediating autophagy in cervical cancer tumorigenesis, we performed cell invasion assay, migration assay, and colony formation assay in vitro and in vivo.The Transwell chamber assay showed that MLLT4-AS1 overexpression significantly inhibited the invasive ability assay of HeLa cells (Fig. 4A).Considering that metastasis is the main cause of death in patients, the effect of MLLT4-AS1 on cervical cancer cells was also investigated.As shown in Fig. 4B, MLLT4-AS1 overexpression significantly inhibited the migration ability of HeLa cells.The overexpression of MLLT4-AS1 decreased the tumor cell growth of HeLa cells in both plate colony formation assay (Fig. 4C).
Then we tried to assess the effect of MLLT4-AS1 on autophagy and tumor growth in vivo.By stripping the tumors from the nude mice, we established xenografts for the different groups after 20 days of treatment (Fig. 4D).Firstly, to confirm the animal model well-constructed, we examined the mRNA expression of MLLT4-AS1 in mice treated with MLLT4-AS1-overexpressed or control cells (Fig. 4E).The tumors that formed in the MLLT4-AS1-overexpressed group were found to be substantially smaller than those in the control group (Fig. 4D, F).Furthermore, higher levels of LC3 puncta (Fig. 4G) were observed in the tumor of mice from the MLLT4-AS1-overexpressed group.And LC3-II levels were significantly induced and p62 levels were decreased in MLLT4-AS1-overexpression xenograft tissues (Fig. 4H).These data suggested that MLLT4-AS1 was involved in the growth of cervical cancer cells, most likely via its activation of ADCD.

Depletion of the lncRNA MLLT4-AS1 attenuates stress-induced autophagy in cervical cancer cells
To identify the essential role of the lncRNA MLLT4-AS1 in basal and stress-induced autophagy, we also performed a loss-of-function assay.The expression of MLLT4-AS1 was knocked down in the unstressed condition and in PP242-treated cells (Fig. 5A,B).MLLT4-AS1 knockdown obviously blocked autophagy induced by PP242, which was shown as the conversion of LC3-I to LC3-II and the degradation of SQSTM1 protein expression (Fig. 5C,D).The level of LC3-II puncta also proved the effect of MLLT4-AS1 knockdown on stressinduced autophagy (Fig. 5E,F).In the presence of chloroquine (autophagy inhibitor, CQ), the LC3-II levels in MLLT4-AS1-overexpressing cells were increased compared with the vector control, which further demonstrated that the enhancement of autophagy flux was almost offset by the inhibition of CQ (Figure S4A).Furthermore, treatment with CQ supported the low level of autophagy flux in MLLT4-AS1 knockdown cells (Figure S4B).These data suggested that MLLT4-AS1 plays an important role in regulating the stress-induced autophagy in cervical cancer cells.

ATG14 is essential for MLLT4-AS1-induced autophagy and ADCD
Next, RNA sequencing was performed to gain deeper insights into the molecular mechanisms underlying MLLT4-AS1-induced autophagy in cervical cancer (Fig. 6A).Probes targeting MLLT4-AS1, divided into odd or even pools, were used to analyze.We identified 593 candidate genes as potential direct downstream targets of MLLT4-AS1 (Fig. 6B).Furtherly, KEGG database was subjected to investigate the differentially accumulated genes.And the genes, which may be associated with autophagy, were selected.Interestingly, ATG14, an autophagy-related mRNA, was identified and directly targeted by MLLT4-AS1.According to the data in Fig. 6C, MLLT4-AS1 was positively associated with ATG14 expression.Then we sought to identify whether ATG14 is crucial for MLLT4-AS1-regulated autophagy.By performing a series of gain-or loss-of-function experiments, we found that inhibition of ATG14 attenuated MLLT4-AS1-induced ADCD of cervical cancer cells (Fig. 6D).Additionally, the suppression of ATG14 eliminated the autophagy flux induced by MLLT4-AS1 in cervical cancer cells (Fig. 6E).

MLLT4-AS1 activates transcription of ATG14 by recruiting MYH-9
It is well known that lncRNAs can directly cooperate with proteins and regulate gene expression via posttranslational regulation.So we performed RNA pull-down followed by mass spectrometry to find out the proteins associated with MLLT4-AS1 (Fig. 7A).Consequently, 1779 potential MLLT4-AS1-interacting proteins were identified (Table 1).Among these proteins, we detected MYH-9, which plays essential roles in autophagy and maintaining cellular mRNA stability.Using MLLT4-AS1 probe, our RNA pull-down assay data found that MYH-9 protein was enriched by MLLT4-AS1 (Fig. 7B,C).Moreover, by performing RIP assay, it was verified that MLLT4-AS1could be precipitated by MYH-9 antibody (Fig. 7D).Our data indicate that MLLT4-AS1 participates with MYH-9 protein to play biology functions.Next, we tried to find out whether MLLT4-AS1could increases ATG14 mRNA stability by binding with MYH-9.As shown in Fig. 7E, the silencing of MYH-9 abrogated the MLLT4-AS1-induced increase in ATG14 mRNA.In addition, RIP assay was performed to determine the role of MYH-9 in mRNA stability of ATG14 induced by MLLT4-AS1.The overexpression of MLLT4-AS1 increased endogenous MYH-9 binding to ATG14 in cervical cancer cells (Fig. 7F, G).On the contrary, knockdown of MLLT4-AS1 exerted the opposite effect in HeLa and Siha cells (Fig. 7H, I).Overall, our results demonstrated that MLLT4-AS1 could increase the mRNA stability of ATG14 though directly binding to MYH-9.

Knockdown of MLLT4-AS1 reverses autophagy in vivo
On basis of our previous observations, further studies should investigate our data in vivo by establishing xenografts in BALB/c nude mouse models.Hela cells with depleted lncRNA MLLT4-AS1(sh-MLLT4-AS1) and the negative control (sh-NC) were bilateral subcutaneous injected respectively (Fig. 8A), and the tumor size in the mice was measured every 2 days (described in detail in the Materials and Methods).It was found that tumor volumes of the sh-MLLT4-AS1 group were substantially smaller than the sh-NC group (Fig. 8A, C).Additionally, we verified that significantly decreased expression of MLLT4-AS1 was observed in sh-MLLT4-AS1 group compared to the control group (Fig. 8B).Our data suggest that MLLT4-AS1 might function as a tumor suppressor in cervical cancer.By using an IHC assay, we tested the hypothesis that MLLT4-AS1could induce the autophagy in vivo.As shown in Fig. 8D, the increased expression levels of SQSTM1 were tested in sh-MLLT4-AS1 group www.nature.com/scientificreports/than the control group.Consistently, sh-MLLT4-AS1 tissue samples showed lower LC3-II and higher p62 protein levels than did the control tissues (Fig. 8E).In addition, the mRNA level and protein expression of ATG14 were downregulated in the sh-MLLT4-AS1 tissues (Fig. 8F, G).Moreover, silencing MLLT4-AS1 decreased endogenous MYH-9 binding to ATG14 in the sh-MLLT4-AS1 tissues (Fig. 8H, I).

Discussion
This study illustrated the importance of lncRNA in stress-induced autophagy in cervical cancer.RNA sequencing data indicated that lncRNA MLLT4-AS1 significantly enriched in MTOR inhibition in cervical cancer.The overexpression of MLLT4-AS1 activates autophagy, induces cell death, and inhibits cervical cancer growth and migration.Gain-and loss-of-function assays showed that the knockdown of MLLT4-AS1 reverses autophagy and ADCD.In addition, ATG14 was verified as a functional target of MLLT4-AS1 during autophagy.Mechanistically, MLLT4-AS1 binding with MYH-9 promotes the translation of ATG14 mRNA, and then leads to autophagy and ADCD (Fig. 9).
Much of the research have examined that lncRNA could affect neighboring genes to regulate cancer initiation and progression 1,19 .MLLT4, as the fusion partner of acute lymphoblastic leukemia (ALL-1) gene, is generally associated with an unfavorable prognosis 20,21 .Our research reveals its antisense transcript, MLLT4-AS1, serves as an upregulated lncRNA in autophagy-induced cervical cancers.Using the UCSC Genome Browser (http:// genome.ucsc.edu/), we analyzed the transcriptional modification region of MLLT4-AS1 and found an enriched H3K27ac binding area upstream at its promoter region.Furthermore, our data suggests that mTOR inhibitor PP242 could transcriptionally activate MLLT4-AS1 expression by inducing H3K27 acetylation.Although the role of MLLT4-AS1 in cancer has not yet been elucidated, our findings confirm that its function in stress-induced autophagy is important.
Accumulating evidence suggests that autophagy is involved in tumor survival under adverse conditions, including nutrient deficiency, stress, chemotherapy, and radiation treatment 22 .It is widely accepted that autophagic activity could regulate many antitumor actions, including chemotherapy, radiotherapy and targetedtherapies 23,24 .Thus, autophagy has been proposed as a potential target in cancer therapy and lncRNA may contribute to these processes.Notably, we found that MLLT4-AS1 was a vital autophagy-regulating lncRNA by inducing autophagy-related cancer death.The molecular mechanism of autophagy involves several conserved ATGs, which have multiple functions in various physiological contexts.Among these genes, ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes 25 .Recent studies have also demonstrated that ATG14 interacts with virus-induced autophagy and immunity 26,27 .Previous reports have shown that ATG14 facilitates cancer development, tumor immunity, cell differentiation, and chemoresistance, including different types of stress 14,26,28,29 .In this research, we proved that MLLT4-AS1 post-transcriptionally regulates the expression of ATG14, indicating the great value of the MLLT4-AS1-ATG14 regulatory pathway in autophagy.
To deeply investigate the mechanism by which MLLT4-AS1 regulates ATG14 protein levels, we screened MLLT4-AS1-interacting proteins and identified myosin-9 (MYH-9).MYH-9 (Myosin Heavy Chain 9), which encodes a conventional non-muscle myosin, has been associated with cytokinesis, cell motility and maintenance of cell shape [30][31][32] .Several studies have indicated that MYH9 favors cancer proliferation, metastasis, invasion and drug resistance 33,34 .Defects in this gene is also related with poor survival in human squamous cell carcinoma, suggesting it acts as a potential tumor suppressor 35 .In the present study, our data revealed that MYH9 acts as an adaptor protein, directly binding with ATG14 mRNA and that MLLT4-AS1 could increase this association.It is plausible that MLLT4-AS1 showed no effect on ATG14 protein stability, which would further support our hypothesis.
Consistent with our in vitro findings, a positive correlation between MLLT4-AS1 and ADCD was confirmed in a subcutaneous mouse model.In addition, MLLT4-AS1 downregulation was significantly associated with both low expression of ATG14 and autophagy flux.These results demonstrate that MLLT4-AS1, which could be significantly induced upon MTOR inhibition, is essential for autophagy-induced tumorigenesis in cancer.Therefore, these findings support the notion that MLLT4-AS1 constitutes an indispensable component of stressinduced autophagy in cervical cancer and may represent a potential therapeutic target.
There are several limitations in our research.First, Hi-Seq sequencing analysis was only performed for HeLa cells.Regarding of this limitation, we validated our findings in both HeLa and SiHa cell lines.In future, it may be  more powerful to include more cervical cancer cells in the sequencing analysis.Second, although we preliminarily proved that the autophagy-related lncRNA MLLT4-AS1 post-transcriptionally regulated the axis of MYH-9/ ATG14/ADCD in cervical cancer, the precise regulatory model and in-depth understanding of the interactions with other cellular factors in the tumor microenvironment will require further study to be fully elucidated.

Conclusion
In conclusion, our study suggested that MLLT4-AS1 plays a crucial role in autophagy-related cell death in cervical cancer by inducing direct regulation of the MLLT4-AS1/MYH-9/ATG14 axis.Mechanistically, PP242-induced stress could enhance MLLT4-AS1 transcription by increasing local H3K27ac modification.The MLLT4-AS1/ MYH-9/ATG14 axis may serve as a prognostic biomarker and promising therapeutic target for cervical cancer survival and progression.Our results provide insight into lncRNA autophagy-regulated therapeutic effects in cancer therapy and a basis for the development of novel and more efficient strategies to treat cervical cancer.

Materials and methods
Cell culture studies HeLa (RRID:CVCL_0030) and SiHa (RRID: CVCL_0032) cells were purchased from American Type Culture Collection (Manassas, VA, USA) and maintained in complete Dulbecco's modified Eagle's medium (Gibco, 10108) supplemented with 10% (vol: vol) fetal bovine serum (FBS; Gibco, 16140071) in a humidified incubator with 5% CO 2 at 37 °C.The details of cell line and mycoplasma-free cells authentications were put in the supporting document 1&2.

Immunoblotting analysis
Cells with different treatments were washed and harvested.And then lyse the cell pellet with lysis buffer and centrifuged at 12,000 × g at 4 °C for 20 min.Transfer the supernatant and mix with sample buffe.The protein samples were boiled and determined.Then loaded up to each well of a of a 15% polyacrylamide gels.And the proteins were transferred to PVDF membrane (Millipore; IEVH08100) according to the manufacturer's instruction.The membranes were blocked with 5% (w/v) bovine serum albumin for 1 h, and then probed with antibodies at 4 °C overnight.Antibodies used in immunoblotting included SQSTM1/P62 (Abcam, ab109012); ATG14 (CST,5504); LC3 (Abcam, ab192890); MYH-9 (Abcam, ab51098); and β-actin (Beijing TDY Biotech CO., TDY166F).Horseradish peroxidase (HRP)-conjugated secondary antibodies were used for detection.

Expression profile analysis of lncRNAs
HeLa cells were treated with PP242 (10 µM) or PBS for 4 h.Three samples were included in each group.Samples were labeled and hybridized on a SurePrint G3 Human Gene Expression Microarray (oebiotech, Shanghai, China; HT2021-22455).The microarray contains 61,760 probes for 21,701 human mRNAs and 10,378 human lncRNAs (reference to databases RefSeq, GenBank, Ensemble, and the Broad Institute).Genespring GX software (Agilent Technologies) was applied to data analyses.Genespring software version 12.0 (Agilent Technologies) was used to quintile normalization and subsequent analysis.Two-fold changes of lncRNA expression were considered differentially in the heatmap.

Cell migration assay
HeLa cells were transfected with MLLT4-AS1, control vector, and PBS treated as a solvate control.The migration ability of HeLa cells was evaluated using the 24-well Transwell system (Corning, USA) according to the

Cell invasion assay
HeLa cells were transfected with MLLT4-AS1, control vector, and PBS treated as a solvate control.The Matrigel matrix (Corning Biocoat; 356234) was diluted and carefully covered the bottom of each Transwell insert (8 μm PET membrane; Corning 3464) for the invasion assays.In this assay, cell samples were placed in the upper chambers with serum free medium.The lower chamber contained 10% FBS.After 24 h, the invasive cells pass though the protein matrix layer and adhere to the bottom of the upper lower membrane.The invasived cells were fixed, stained and photographed.Cells in five fields in each well were captured under a microscope (magnification × 200).

RNA immunoprecipitation (RIP) assay
RIP assay was conducted using a Magna RIP RNA-binding protein immunoprecipitation (IP) kit (Millipore, USA), according to the manufacturer's instructions.Cells in exponential growth period were treated as different required.Harvest cells and resuspend in nuclear isolation buffer on ice.Nuclei were pelleted and resuspended in RIP buffer.Then the suspended nuclei were divided into two parts (for mock and IP).Chromatin were sheared by homogenizer, and the debris were centrifuged and separated.Add antibody to the protein and incubate gently overnight at 4℃.And then protein beads were incubated and resuspended in RIP buffer.The coprecipitated RNAs and beads were seperated by resuspending in TRIzol RNA extraction reagent.Elute RNA with nuclease-free water.The isolated proteins can be analyzed using western blot analysis, and the isolated RNAs were detected by qRT-PCR.

Chromatin immunoprecipitation (ChIP) assay
ChIP assay was implemented using a Simple ChIP enzymatic ChIP kit (CST, 56383).Cells were incubated with formaldehyde to crosslink the protein and DNA complexes.Quench the formaldehyde, and the cells were resuspended in Lysis Buffer.Then chromatin was sheared to about 1 kb length by sonicating samples.Collect the sample and dilute it by dilution Buffer.The samples were incubated with antibodies and added with Streptavidin beads.Then the mixture was washed with wash buffer, and suspended with Chelating Resin Solution.The DNA samples were purified, concentrated, and resuspended in deionized water.The isolated DNAs were quantitated by qRT-PCR.The specific antibodies used in this study were as follows: H3K27ac antibody (Abcam, ab4729); the negative control IgG antibody (Millipore, 12-371).

RNA pull-down and mass spectrometry
lncRNA MLLT4-AS1, which was labeled with biotin, was transcribed and purified by RNeasy Plus mini kit (QIAGEN, 74134) according to the manufacturer's instructions.The products were treated with DNase I (NEB, M0303S).To verify whether the RNAs were biotinylated and transcribed correctly, biotin-HRP Northern blot was used to this assay according to manufacturer's manual (NorthernMax kit, Ambion).Then biotinylated RNAs were heated and supplied with RNA structure buffer.Nuclei were centrifuged and resuspended in RIP buffer (Roche Complete Protease Inhibitor Cocktail Tablets)).Nucleus were resuspended and mechanically sheared by homogenizer.Nuclear extracts in RIP buffer were added with folded RNA.And then the washed Streptavidin agarose beads (Invitrogen) were mixed with each mixture, incubated and washed in Handee spin columns (Pierce).The isolated proteins were separated by electrophoresis, and identified with mass spectrometry (Shanghai Bioprofi-leTechnology, Shanghai, China).Proteins were analyzed with MaxQuant 1.6.1.0(Germany), and referred to the human RefSeq protein database (National Center for Biotechnology Information).

Colony formation assay
HeLa cells were transfected with MLLT4-AS1, control vector, and PBS treated as solvate control.For each sample, 1 × 10 3 cells with different treatments were cultured at 6-well plates in complete medium for 7 days.The colonies, analysis revealed that the tumors developed from sh-MLLT4-AS1 cells displayed higher SQSTM1 staining than did the sh-NC group.(E) Western blot showed that expression of LC3I/II and SQSTM1 in sh-MLLT4-AS1 and control tissues.(F) The mRNA expression of ATG14 was decreased in sh-MLLT4-AS1 tissues.(G) Immunohistochemistry analysis revealed that the tumors developed from sh-MLLT4-AS1 cells displayed lower ATG14 staining than did the sh-NC group.(H) RIP was performed using anti-myosin-9 and control IgG antibodies, followed by quantitative real-time PCR to examine the enrichment of MLLT4-AS1 and U6 in sh-MLLT4-AS1 and control tissues.U6 served as a negative control.(n = 20, Student's t-test, ***p < 0.01).
neutral buffered formalin, and dehydrated in ethanol.And hybridization of samples was conducted in a hybridization buffer-covered slide incubation chamber.Then wash the samples gently with hybridization buffer, and counterstained with mounting medium containing 1.5 µg ml-1 4′,6-diamidino-2-phenylindole (DAPI).Finally, the samples were counted and analyzed by a fluorescence microscopy.The sequence used for the ZNF649-AS1 probe was as follows: 5′-CCA GCG GAA AGG AAT CAC CCT GGA AG-3′.

Immunohistochemistry (IHC) analysis
Immunohistochemistry staining was performed as described previously 5 .Briefly, samples were collected from xenograft tumor tissues and applied primary antibody targeting LC3 (1:500) (Abcam, ab192890).The color of antibody staining in the tissue sections was observed under microscopy.

Xenograft assays
All animal studies were approved by the Xi'an Jiao Tong University Animal Care and Use Committee.Four-weekold BALB/c nude female mice were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China) and housed in the SPF Laboratory Animal room.Notably, counted differently treated cells and prepare the cell suspension for final concentration (5 × 106 cells/100 μl).200 μl cell suspension was injected into two flanks of one mouse (left: vector/Sh-NC; right: MLLT4-AS1/Sh-MLLT4-AS1) each.The tumor volumes and weights were measured every 2 days using a vernier calipers.After several weeks, the tumor volumes were calculated according to the following formula: tumor volume = (length × width × width)/2.

Figure 1 .
Figure 1.Identification of MLLT4-AS1 as an autophagy-related lncRNA.(A) PP242 induced autophagy in Hela and Siha cells.The cells were treated withPP242 (10 µM) for 4 h and the conversion of LC3-I to LC3-II and SQSTM1 degradation were then analyzed by western blotting (n = 3) using ACTB/β-actin as the loading control.(B) Gene expression variations between Hela and PP242-treated Hela cells were screened by RNA sequencing and presented as volcano plot.(C) The heatmap shows the lncRNAs with at least two-fold change in expression between the PP242-treated groups and controls.(D) Genomic structure of lncRNA MLLT4-AS1.(E) Real-time PCR analysis of mRNA expression of MLLT4-AS1 in Hela and Siha cells, treated with PP242 (10 µM) for 4 h.

Figure 2 .
Figure 2. ZNNT1 is an intranuclear lncRNA and facilitated the autophagy in cervical cancer cells.(A) RNA fluorescence in situ hybridization (RNA-FISH) verified that MLLT4-AS1 was mainly distributed in cytoplasm of Hela cells.(B) Cellular fractionation PCR showed that MLLT4-AS1 was mainly distributed in the cytoplasm section.(C) Western blot showed that MLLT4-AS1 overexpression increased the conversion of LC3-I to LC3-II and the degradation of SQSTM1.(D) Overexpression of MLLT4-AS1 increased the numbers of LC3-II puncta in cervical cancer cells.(E) Western blot analysis of the LC3 and SQSTM1 protein levels in MLLT4-AS1-knock down and control cells that transfected with MLLT4-AS1 siRNA for 24 h.(F) Downexpression of MLLT4-AS1 decreased the numbers of LC3-II puncta in cervical cancer cells.Scale bar: 10 μm.

Figure 4 .
Figure 4. MLLT4-AS1 inhibits tumorigenesis and the migration ability of cervical cancer.(A) MLLT4-AS1 overexpression inhibits the invasive ability of Hela cells.A transwell assay was performed to analyze the effect of MLLT4-AS1-overexpressing on the invasive ability of Hela cells.The invasive cells in 10 representative scans were counted and analyzed.(*)p < 0.05, (***)p < 0.01, scale bar: 100 μm.(B) MLLT4-AS1 overexpression inhibits the migrative ability of Hela cells.A transwell assay was performed to analyze the effect of MLLT4-AS1-overexpressing on the migrative ability of Hela cells.The migrative cells in 10 representative scans were counted and analyzed.(*)p < 0.05, (***)p < 0.01, scale bar: 100 μm.(C) The in vitro proliferative ability of Hela cells was significantly decreased in the MLLT4-AS1 overexpressing cells compared with the mock-transfected control cells, as demonstrated by a plate colony formation assay.The number of colonies per well were counted and analyzed.(*)p < 0.05, (***)p < 0.01, n = 3. (D) Hela cells with MLLT4-AS1-overexpressing (right side) or not (left side) were injected subcutaneously into BALB/c female mice (n = 5).(E) Real-time PCR analysis of mRNA expression of MLLT4-AS1 in MLLT4-AS1-overexpressed xenograft tissues compared to controlled tissues.(F) Tumor volume was measured every two or three days.And calculated as: (length × width × width)/2.Each data point represented the mean ± SD. (*)p < 0.05.(G) GFP staining showed that LC3 puncta was suppressed in MLLT4-AS1-overexpressed xenograft tissues compared to controlled tissues.(H) Western blot analysis of the LC3 and SQSTM1 protein levels in MLLT4-AS1-overexpressed xenograft tissues compared to controlled tissues.

Figure 6 .
Figure 6.MLLT4-AS1 transcriptionally activates ATG14 expression.(A) Venn diagram of MLLT4-AS1 targets selection.Differential genes were selected by comparing (WT) to MLLT4-AS1 overexpression (OE) cells.MLLT4-AS1 DNA binding sites were selected by merging odd and even pool of ChIRP-seq.RNA-DNA binding in-2000-200 bp to transcriptional start site (TSS) were selected.(B) KEGG analysis of the 593 candidate genes.(C) The mRNA expression of ATG14 was increased in MLLT4-AS1-overexpressing cervical cancer cells.(D) MMT assay revealed that suppression of ATG14 abolished the ADCD induced by MLLT4-AS1 in cervical cancer cells.(E) Western blot showed that suppression of ATG14 abolished the autophagy activity induced by MLLT4-AS1 in cervical cancer cells.The data are shown as the mean ± SD of three independent experiments.(*) p < 0.05, (***) p < 0.01, n = 3.

Figure 7 .
Figure 7. MLLT4-AS1 interacts with myosin-9.(A) Identification of proteins associated with MLLT4-AS1.Left: silver staining of RNA pull-down proteins.Whole cell lysates were incubated with in vitro transcribed RNA of over-expressed cells.Right: Representative image of gel band analysis after silver staining by mass spectrometry.(B, C) RNA pull-down assay showed that myosin-9 protein was directly enriched by MLLT4-AS1 probe in cervical cancer cells.(D) RIP was performed using anti-myosin-9 and control IgG antibodies, followed by quantitative real-time PCR to examine the enrichment of MLLT4-AS1 and U6.U6 served as a negative control.(E) Quantitative real-time PCR showed that MLLT4-AS1 upregulated ATG14 expression; however, this upregulation was significantly reversed by co-transfection of si-myosin-9(Si-MYH-9).(F, G) The endogenous myosin-9 binding to ATG14 mRNA was increased by overexpression of MLLT4-AS1 in cervical cancer cells, while it was decreased by knockdown of myosin-9.(H, I) The endogenous myosin-9 binding to ATG14 mRNA was decreased by downexpression of MLLT4-AS1 in cervical cancer cells, while it was decreased by knockdown of myosin-9.The data are shown as the mean ± SD of three independent experiments.(*) p < 0.05, (***) p < 0.01, n = 3.

Figure 9 .
Figure 9.A scheme of the proposed mechanisms.Stress treatment increases the H3K27ac enrichment at the MLLT4-AS1 promoter region, inducing upregulation of MLLT4-AS1, which thereby increases ATG14 expression by guiding myosin-9 to activate its transcription.Then, increased ATG14 protein induced autophagy and ADCD.

Table 1 .
MS result of MLLT4-AS1 RNA pull-down.MS, mass spectrometry; MW, Molecular weight [kDa]; PI, isoelectric point.'s instructions.In this assay, cell suspensions were placed in the upper compartments with serum free medium.The lower chambers contained 10% FBS.After 12 h, the migratory cells pass though polycarbonate membrane to the lower compartment.The migratory cells were fixed, stained and photographed.Cells in five fields in each well were captured under a microscope (magnification × 200). manufacturer